Paclitaxel production by actinomycetes

ABSTRACT

The present invention related to microorganisms producing taxanes, such as paclitaxel or related taxanes, to the use of said microorganisms for the production of taxanes, and to the procedure for the isolation of said microorganisms from plants. In particular, the present invention relates to microorganisms of the Actinomycetes group producing taxanes as well as to the biologically pure cultures of a strain of the Actinomycetes group which produces taxanes. The microorganisms of Actinomycetes group are preferably belonging to the suprageneric groups of Streptomycetes, Actinoplanetes, Maduromycetes, Thermomonosporas or Nocardioforms, more preferably they are belonging to genus Streptomyces, Actinoplanes, Nocardiopsis, Micromonospora, Actinomadura or Kitasatosporia, still more preferably to the genus Kitasatosporia. A particularly preferred microorganism is Kitasatosporia sp. CECT 4991.

This application is a National Stage Application of InternationalApplication Serial No. PCT/EP99/00417, filed on Jan. 21, 1999, andclaims priority under 35 U.S.C. §119 to GB 9803628.8, filed on Feb. 20,1998, and GB 9809767.8, filed on May 7, 1998.

The present invention relates to microorganisms producing a taxane, tothe use of said microorganisms for the production of a taxane, and tothe procedure for the isolation of said microorganisms from plants.

Paclitaxel, a diterpene which is of the chemical structural formula:

shows significant properties of promoting the polymerization of tubulinand inhibiting the depolymerization of microtubules. For these reasons,paclitaxel is a valuable antileukemic and antitumor agent and is thesubject of increasing research. Paclitaxel has been identified in 1971(Wani M C et al, J. Amer. Chem. Soc. 1971, 93, 2325-2327) by isolatingit from the bark of Taxus brevifolia (Pacific yew). The low yield of theisolation of paclitaxel (around 0.007%) from this source and theconcerns about the survival of Taxus brevifolia, which is a rather slowgrowing plant, raised by its massive collection have pushed the researchfor alternative paclitaxel production methods.

Isolation of paclitaxel from a renewable source such as the needles andthe twigs of Taxus species has proven possible although with a yieldcomparable, if not lower, than that obtainable from the bark of Taxusbrevifolia.

The total chemical synthesis of paclitaxel has been described by Nicolauet al., Nature 1994, 367, 630-634, nevertheless the complexity and lowyield of this method have prevented its industrial scale-up.

Semisynthesis of paclitaxel from baccatin III or 10-deacetylbaccatinIII, has proved to be a much better option because of the use of arenewable source such as the needles of the European yew Taxus baccatato obtain the precursors.

Several documents on tissue culture of plants of the genus Taxus havebeen published in recent years, however this procedure is limited by theintrinsic technical difficulties of cell culture on an industrial scale.

Recently, patents and patent applications for the production ofpaclitaxel from the fermentation of fungi and bacteria isolated fromsamples of Taxus have been published (WO 93/21338, U.S. Pat. No.5,322,779, WO 95/04154, U.S. Pat. No. 5,561,055 and WO 96/32490). Theseconfirm that there is a need for improved methods for the production ofpaclitaxel on industrial scale.

In this specification the used classification rules are described inBergey's Manual of Systematic Bacteriology, vol. 4, 1989.

We have surprisingly found a new taxanes producing microorganism of theActinomycetes group, which is particularly well suited for an industrialfermentation process for the production of a taxane such as paclitaxeland related taxanes. For instance, compared to the fungi of the priorart, the microorganisms of the present invention can be more easilygenetically modified to enhance taxane production and have a shorterfermentation time.

The present invention relates to microorganisms of the Actinomycetesgroup which produce a taxane, such as paclitaxel or related taxanes, aswell as to the biologically pure cultures of a strain of theActinomycetes group which produce a taxane such as paclitaxel or relatedtaxanes.

The microorganisms of the invention preferably belong to thesuprageneric groups of Streptomycetes, Actinoplanetes, Maduromycetes,Thermomonosporas or Nocardioforms, more preferably they belong to thegenus Streptomyces, Actinoplanes, Nocardiopsis, Micromonospora,Actinomadura or Kitasatosporia and still more preferably to the genusKitasatosporia.

A preferred strain according to the present invention is aKitasatosporia sp. deposited under the Budapest treaty at the ColleccionEspañola De Cultivos Tipo, (Biological Science Faculty, University ofValencia, 46100 Burjasot (Valencia), Spain) on Jan. 19, 1998 under thedeposit number CECT 4991, and it was isolated from a Taxus baccatasample and proved to produce paclitaxel or other related taxanes.Therefore, the present invention more preferably relates to thebiologically pure cultures of the genus Kitasatosporia having all theidentifiying characteristics of Kitasatosporia sp. CECT 4991.

Moreover, the present invention provides a procedure for the isolationof microorganisms of the Actinomycetes group which produce a taxane,such as paclitaxel or related taxanes, from plants, including plants ofthe Taxus genus and related genera, collected from various geographicalarea.

Preferably, the isolation procedure of microorganisms according to thepresent invention is carried out on plants selected from plants of Taxusgenus or related genera, more preferably Taxus baccata, Taxusbrevifolia, or Torreja californica.

According to the present invention, the procedure for the isolation ofmicroorganisms of the Actinomycetes group producing a taxane, such aspaclitaxel or related taxanes, may be carried out, for example, bysterilizing each part of the plant (roots, bark, twigs, needles, stems)with a sterilizing agent, preferably selected from sodium hypochlorite,propylenoxid, a solution of dodecylcarbamylmethyl-benzyldimethylammoniumcloride, triethilenglycol sodium nitrite and UV light, by cutting thedifferent parts of the plant in small fragments with a sterile blade andby placing these small fragments on agar culture media in Petri platesor, preferably, by homogenizing the sterilized parts as above, with ablender at room temperature; the resultant homogenate, reduced to powdermay be then transferred on agar culture media in Petri plates using amodified Andersen's sampler. The microorganism colonies grown on theagar plates are then removed and transferred to plates containing wateragar media until a pure culture is obtained.

The colonies were grown in liquid media suitable for growth and taxaneproduction, such as conditions suitable for paclitaxel production. Theextracts and the fractions obtained from the production cultures can betested to determine the presence of taxanes, such as paclitaxel orrelated taxanes, using suitable assays.

In the more preferred procedure for the isolation of the microorganismsof the Actinomycetes group from Taxus and related plants, the powderedsamples obtained as above described are introduced into a modifiedAndersen's sampler, an apparatus used to analyze the microorganismspopulation in air samples. Andersen's sampler is described in detail in:A. Andersen, Journal of Bacteriology 1958, 76 (5), 471-484. This sampleris modified by associating it with a sedimentation chamber in which thepowdered samples are introduced. The sedimentation chamber allowsisolation of microorganisms present in low percentage from soil andvegetable powdered samples. It consists of a cylindric box made ofplexiglass connected to the sampler by a rubber tubing. Platescontaining different isolation media were placed in the sampler and thepowdered samples were drawn. Depending on their aerodynamic dimensions,the microorganisms present in the powdered samples were collected onplates containing different isolation media. The plates are thenremoved, incubated and examined. The colonies of microorganisms of theActinomycetes group grown on the agar plates were picked up andtransferred on water agar media in Petri plates until a pure culture wasobtained. This method enables the isolation of microorganisms of theActinomycetes group well separated from fungi and other bacteria.

A further object of the invention is the use of said microorganisms ofthe Actinomycetes group for the production of a taxane, such aspaclitaxel or related taxanes, which comprises the steps of

a) culturing a microorganism of the Actinomycetes group producing thetaxanes in a liquid media suitable for its growth and production

b) recovering the taxanes produced.

Thus the invention provides a biologically pure culture of amicroorganism of the invention. Such a culture may be substantially freeof other microorganisms. The term “related taxanes” is intended toinclude paclitaxel and all taxane skeleton-containing compounds, whichincludes taxane skeleton-containing compounds thought to be precursorsin the biosynthesis, for example baccatin III, 10-deacetyl baccatin III,cephalomannine, 10-daecetylcephalomannine. The term also includesdeacetyltaxol, 7-xylosyltaxol, 7-epi-10-deacetyltaxol,7-xylosyl-10-deacetyltaxol, 7-epi-taxol and 7-epi-10-deacetylbaccatinIII.

The term “related taxanes” also includes taxanes which are recognised byan antibody produced to paclitaxel, 10-deacetyltaxol, 7-xylosyltaxol,7-epi-10-deacetyltaxol, 7-xylosyl-10-deacetyltaxol, 7-epi-taxol,7-epi-10-deacetylbaccatin III, baccatin III, 10-deacetyl baccatin III,cephalomannine or 10-deacetylcephalornannine. Such antibodies can bepolyclonal antibodies or monoclonal antibodies. Polyclonal antibodiescan be produced using standard techniques, such as immunising a hostanimal with paclitaxel, 10-deacetyltaxol, 7-xylosyltaxol,7-epi-10-deacetyltaxol, 7-xylosyl-10-deacetyltaxol, 7-epi-taxol,7-epi-10-deacetylbaccatin III, baccatin III, 10-deacetyl baccatin III,cephalomannine or 10-deacetylcephalomannine and obtaining the polyclonalantibodies from the animal, for example from the serum. Monoclonalantibodies can also be produced by standard techniques, such as themethod of producing monoclonal antibodies of Kohler and Milstein (Nature256, 495-497 (1975)). To produce such antibodies using the Kohler andMilstein method paclitaxel, 10 deacetyltaxol, 7-xylosyltaxol,7-epi-10-deacetyltaxol, 7-xylosyl-10-deacetyltaxol, 7-epi-taxol,7-epi-10-deacetylbaccatin III, baccatin III, 10-deacetyl baccatin III,cephalomannine or 10-deacetylcephalomannine would be used to immunisethe animal. In particular, “related taxanes” includes those recognisedby the antibody R4, 8A10, 3C6 or 3H5. These antibodies are available inkits from Hawaii Biotechnology Group Inc. Preferred “related taxanes”are those which have an IC50 (concentration of taxane required toinhibit the binding of the antibody to solid phase antigen, which may bepaclitaxel or baccatin III, by 50% in a competitive immunoassay) of lessthan 500 nM, less than 250 nM, less than 100 nM, less than 50 nM, orless than 10 nM, as measured using a competitive immunoassay kit, suchas from Hawaii Biotechnology Group Inc.

To grow the isolated colonies the following culture media can be used:water agar, Czapek agar, ISP medium 2 (International StreptomycesProject, Shirling E. B. and Gottlieb D., Int. J. Syst. Bacteriol. 1966,16, 313-340) adding antifungal and antibacterial substances. Themicroorganism colonies grown on the agar plates are removed andtransferred to water agar media in Petri plates until a pure culture isobtained. After purification, the colonies of the Actinomycetes groupproducing taxanes, such as paclitaxel or related taxanes, aretransferred into tube of ISP medium 3.

Each isolated microorganism can be stored either in glycerol at −80° C.or lyophilized. A suitable medium for culturing on organism of theinvention may be one which comprises an assimilable carbon source, anassimilable nitrogen source and inorganic salts.

Each microorganism of the Actinomycetes group producing taxanes, such aspaclitaxel and related taxanes, may be cultured into liquid seed media,containing complex carbon and nitrogen sources, in Erlenmyer flasks andincubated on a shaker at temperatures ranging from 22° to 30° C. and atagitation rates from 150 to 250 rpm for 24-48 hours. At the end ofincubation, the seed culture is inoculated into different productionmedia containing complex substances such as carbon sources (starch,dextrin, morsuit), nitrogen sources (corn-steep liquor, soybean meal,caseine, soytone, yeast extract), ammonium salts (ammonium sulfate,ammonium chloride) and mineral salts; calcium carbonate and potassiumphosphate can be useful. The temperature of incubation can range from22° to 32° C. and the agitation rate from 150 to 250 rpm. Afterinoculation, the fermentation production media are incubated over aperiod of 2 to 8 days.

After the required incubation time, the cultures are centrifuged and thepellet separated from the supernatant. The supernatant of the culturecan be extracted with an organic solvent unmixable with water, forexample dichloromethane.

Preferably the supernatant of the culture is loaded onto a pre-packeddiatomaceous earth column. This material allows efficient liquid-liquidextraction of lipophilic compounds from aqueous solutions withoutemulsion problems. Taxanes, such as paclitaxel and other taxanes can berecovered from the column with various organic solvents.

The mycelium cake of the culture can be extracted by homogenizing itwith an organic solvent, for example methanol.

Preferably, after homogenizing the mycelium of the culture with asolvent, for example methanol, the pellet is removed by centrifugation,while the solvent extract is dried in vacuo, reconstituted with a smallvolume of methanol and then with water.

This solution can be loaded onto pre-packed diatomaceous earth columnsand treated as the supernatant of the culture.

The fermentation of the isolated microorganisms can be scaled up usingfermenter tanks. For instance in order to prepare the inoculum for a 500L fermenter, the actinomycetes can be cultured in liquid seed media,containing complex substances such as carbon sources (starch, dextrin,morsuit), nitrogen sources (corn-steep liquor, soybean meal, caseine,soytone, yeast extract), ammonium salts (ammonium sulfate, ammoniumchloride) and mineral salts; calcium carbonate and potassium phosphatecan also be useful.

The temperature of incubation can range from 22° to 32° C. and theagitation rate from 150 to 250 rpm for 24-48 hours.

At the end of incubation (24 to 48 hours) the seed culture istransferred into an intermediate fermenter containing the same liquidmedium as previously described and incubated for 24 to 48 hours at atemperature ranging from 22° to 32° C., at an agitation rate rangingfrom 150 to 250 rpm and with an appropriate air flow rate, for instance68 nL/min for 24-48 hours. Finally a portion of this culture istransferred to the final tank, for example a 500 L fermenter, with amedium containing complex substances such as carbon sources (starch,dextrin, morsuit), nitrogen sources (corn-steep liquor, soybean meal,caseine, soytone, yeast extract), ammonium salts (ammonium sulfate,ammonium chloride) and mineral salts, calcium carbonate or potassiumphosphate. After a fermentation period ranging between 96 and 176 hours,the mycelium is removed from the culture by centrifugation and the clearsupernatant applied onto a column of chromatographic resin, for instanceAmberlite XAD-2.

After the adsorption, the resin can be eluted with solvents, for exampleethanol and after removal of the organic solvent under reduced pressurethe fractions eluted from the column are extracted with organic solventsunmixable with water such as dichloremethane.

The solvent extract can be purified using reversed-phase C18 columnsthat are eluted using a step gradient of an alcohol, for examplemethanol, in water.

A further purification of paclitaxel can be achieved by means of otherschromatographic procedures, for example preparative HPLC on RP18 orpentafluorophenyl columns. The extracts and the fractions obtained withthe described procedures can be tested to determine the amounts oftaxane, such as paclitaxel or related taxanes using suitable assays.

Such an assay may comprise recognition of the taxane by an antibody to ataxane, such as an antibody to paclitaxel, 10 deacetyltaxol,7-xylosyltaxol, 7-epi-10-deacetyltaxol, 7-xylosyl-10-deacetyltaxol,7-epi-taxol, 7-epi-10-deacetylbaccatin III, baccatin III, 10-deacetylbaccatin III, cephalomannine or 10-deacetylcephalomannine.

Preferably two enzyme immunoassay kits (Indirect Competitive InhibitionEnzyme Immunoassay—CIEIA) employing, respectively, a polyclonal antibodyagainst a taxane skeleton-containing compound and a monoclonal antibodyagainst paclitaxel, are used. Immunoassays and antibodies for paclitaxelcan be obtained from Hawaii Biotechnology Group Inc.

Extracts may be analyzed by HPLC, for example on a reversed-phase C18column using an acetonitrile—water gradient and using a standard curveof paclitaxel concentration versus peak area to quantify paclitaxel insamples.

A further evidence for production of paclitaxel by the isolated strainscan be obtained in feeding experiments with radioactive precursors forexample with L-[2,3,4,5,6-³H]phenylalanine and ³H-Baccatin and alsoanalytically by liquid chromatography coupled to mass spectroscopy(LC-MS) of the paclitaxel isolated from the cultures. The isolatedmicroorganisms of the Actinomycetes group producing a taxane, such aspaclitaxel or related taxanes, can be genetically modified, for exampleby random mutagenesis or biosynthetic gene manipulation, to enhancetaxane production.

The taxane produced by the microorganisms of the invention may act asantitumour agents. A human or animal suffering from a tumor may thus betreated by a method which comprises the administration thereto of aneffective amount of such a taxane. The condition of the human or animalmay thereby be improved.

Examples of tumors that can be treated are sarcomas, carcinomas,lymphomas, neuroblastomas, melanomas, myelomas, Wilms tumor, leukemiasand adenocarcinomas. The taxane can be used to treat ovarian cancer,platinum-resistant ovarian cancer, metastatic breast cancer, non-smallcell lung cancer, and head and neck cancer. The taxane may be used toproduce a pharmaceutical composition which comprises, as activeingredient, the taxane and a pharmaceutically acceptable carrier ordiluent. This composition is usually prepared following conventionalmethods and is administered in a pharmaceutically suitable form.

Administration can be made by any of the accepted ways foradministration of antitumor agents such as intravenous, intramuscular orsubcutaneous injection or topical application. For systemic injectionthe active compound may be, for example, dissolved in a vehicleconsisting of a mixture of polyoxyethlated castor oil (Chremophor EL)50% and ethanol 50% and then diluted with glucose 5% solution at thedesired concentration, or in other pharmaceutically suitable carriers.

The amount of the active compound administered depends on the treatedsubject, age, weight, sex etc., and the severity of the affliction. Themethod of administration depends on the judgement of the prescribingphysician. A suitable dosage for an average 70 kg person may range fromabout 0.01 g to about 1 g per day.

The following Examples illustrate the invention:

Table 1: this table reports the plants from which samples werecollected, the procedures for the isolation of microorganism of theActinomycetes group producing taxanes, such as paclitaxel and relatedtaxanes and the genus of the isolated microorganisms of theActinomycetes group, as described in Examples 1 to 9;

Table 2: cultural and morphological characteristics on different culturemedia of strain Kitasatosporia sp. CECT 4991.

Table 3: Carbohydrate utilization by strain Kitasatosporia sp. CECT4991.

Table 4: Enzymatic activities of strain Kitasatosporia sp. CECT 4991.

Table 5: Purification procedure of the extract obtained from the cultureof strain Kitasatosporia sp. CECT 4991 incubated withL-[2,3,4,5,6-³H]phenylalanine

EXAMPLES 1-9

Isolation of Microorganisms of the Actinomycetes Group ProducingTaxanes, Such as Paclitaxel and Related Taxanes

With the method of the invention, 70 different microorganisms of theActinomycetes group were isolated and demonstrated to produce paclitaxelor related taxanes according to the above described enzyme immunoassay.

A series of nine examples are reported below. The detailed informationof each isolation related to the plants, to methods of pretreatment, toisolation media and to isolated actinomycetales are shown in the table1.

The surface of each part of the plant (roots, bark, twigs, needles,stems) was sterilized with either sodium hypochlorite or propylenoxid ora solution of dodecylcarbamylmethyl-benzyldimethylammonium chloridetriethilenglycol sodium nitrite (IPIT) or UV light. The sample washomogenized with a blender at room temperature, reduced to powder andintroduced into a sedimentation chamber connected to an Andersen'ssampler.

Andersen's sampler is described in detail in: A. Andersen, Journal ofBacteriology 1958, 76 (5), 471-484. The sampler used for the presentinvention has been modified by associating it with a sedimentationchamber in which the powdered samples are introduced. The sedimentationchamber consists of a cylindric box made of plexiglass connected to thesampler by a rubber tubing. The powdered sample was introduced in thesedimentation chamber by a hole situated in the upper side. The samplewas then dispersed by the action of a fan placed in the chamberoperating for 30 seconds. After 10 minutes and then again after one hourof sedimentation, the sample was drawn into the Andersen's sampler andcollected on the agar plates placed in the instrument. The platescontaining different isolation media were then removed, inverted intheir covers, incubated at 28° C. and examined on a day-to-day basis.The colonies of microorganisms of the Actinomycetes group grown on theagar plates were removed and transferred to water agar until a pureculture was obtained. After purification, the microorganisms of theActinomycetes group producing paclitaxel or related taxanes weretransferred into tubes of ISP medium 3.

The strain Kitasatosporia sp. CECT 4991 isolated in Example 3 wasfurther characterized as described in the following examples.

EXAMPLE 10

Characterization of the Strain Kitasatosporia sp. CECT 4991 Culture andMorphological Characteristics

Culture characteristics of the strain were determined after 7, 14 and 21days incubation on the following culture media: 2.5% water agar, yeastextract-malt extract agar (ISP medium 2), oatmeal agar (ISP medium 3),inorganic salts-starch agar (ISP medium 4), glycerol asparagine agar(ISP medium 5), tyrosine agar (ISP medium 7), Nutrient agar, Bennett'sagar and Bacto Czapek solution agar. The culture features of the strainKitasatosporia sp. CECT 4991 are shown in Table 2. The strain grows wellon all organic and synthetic media and the colonies are covered withgray aerial mycelium and spores. Absence of melanoid pigment on ISPmedium 7. Morphological observations were assessed by light and scanningelectron microscopy on cultures grown at 27° C. for 14 days on ISPmedium 3, Nutrient agar, Czapek agar and 2.5% water agar. The substratemycelium is well developed with hyphae densely branched, 0.5-0.8 μm indiameter. Aerial hyphae bear long spore chains of more than 20 spores.The spores are globose, oblong of irregular length (0.8-1.8×0.8-1.2 μm)with smooth surface. Spore chains are long, straight or slightlyflexuous unbranched or poorly irregularly branched.

Physiological Tests

Carbohydrate utilization was carried out by the API 50 CH system(BioMèrieux SA) using as inoculum a 3% agar medium (yeast nitrogen base)neutralized with 20% (v/v) 1% K₂HPO₄ solution. The results aresummarized in Table 3. Enzymatic activities were carried out by the APIZYM system (BioMerieux SA ). Enzymatic activities of the strain areshown in Table 4.

Cell Chemistry

Determination of the cell-wall composition, including diaminopimelicacid isomers and sugars, was carried out according to Becker et al,Appl. Microbiol. 1965, 13, 236-243. Cell wall containingmeso-diaminopimelic acid and whole-cell hydrolysates is characterized bythe presence of galactose and by the absence of madurose, arabinose andxylose. On the basis of that, the cell wall analysis was repeatedaccording to Omura et al, J. Antibiotic, 1981, 34, 1633-1634. Theanalysis of the cell wall showed L-diaminopimelic acid in aerial sporesand meso-diaminopimelic acid in vegetative mycelia. These resultssuggested that the strain CECT 4991 belongs to the genus Kitasatosporia.

EXAMPLE 11

Flask Fermentation of the Strain Kitasatosporia sp. CECT 4991

A frozen vial of the strain Kitasatosporia sp. CECT 4991 was defrostedand transferred in 300 mL Erlenmyer flask containing 30 mL of thefollowing liquid seed medium (VS): corn steep liquor 10 g/L, caseine 10g/L, dextrin 20 g/L, (NH₄)₂SO₄ g/L, CaCO₃ 5 g/L, K₂HPO₄ 0.1 g/L,deionized water to 1000 mL. The flask was incubated under aerobiccondition for 48 hours at 28° C., 250 rpm, 70% humidity. At completionof the incubation, 2 mL aliquot was used to inoculate aseptically a 300mL Erlenmyer flask containing 50 mL of the following liquid productivemedium (PT): morsuit 25 g/L, dextrin 10 g/L, soytone 15 g/L, MOPS 5 g/L,K₂HPO₄ 0.5 g/L, FeSO₄.7H₂O 10 mg/l, MnSO₄.H₂O, 10 mg/L, ZnSO₄.7H₂O 10mg/L, MgSO₄.7H₂O 10 mg/L. The flask was incubated under aerobiccondition for 168 hours at 28° C., 250 rpm, 70% humidity.

Extraction of Culture

After the required 168 hours incubation time, the culture wascentrifuged at 5000 g for 20 min and the pellet set apart for a separateextraction while 20 mL of the culture supernatant were loaded onto apre-packed Extrelut 20 (diatomaceous earth) column (E. Merck, Germany).

After the application of the culture, the column was washed withn-hexane (120 mL) to remove the fatty components of the culture and thenwith dichloromethane (80 mL) to recover paclitaxel and other taxanesfrom the column. The dichloromethane extract was then brought to drynessto be tested by the immunoenzymatic assay (CIEIA). The mycelium cake ofthe culture was extracted by homogenizing it with 10 mL of methanol.After separating the pellet by centrifugation at 5000 g for 30 min., thesupernatant was dried in vacuo, reconstituted in methanol (500 μL) andtaken to a final volume of 20 mL with water. This solution was thentreated as the supernatant.

Indirect Competitive Inhibition Enzyme Immunoassay (CIEIA)

The dried dichloromethane extracts were dissolved in 100 μL of phosphatebuffered saline (PBS ) containing 0.25% (w/v) BSA, 0.05% Tween 20 and20% methanol (PBS-T-M) to obtain 200×concentrated samples.

The suspended extracts were serially diluted in PBS-T-M and tested todetermine the amounts of taxanes and paclitaxel using two kinds ofenzyme immunoassay kits employing a polyclonal antibody against a taxaneskeleton-containing compound and a monoclonal antibody againstpaclitaxel, respectively.

All samples tested by CIEIA were run in duplicate and the mean result ofeach set of replicates was calculated.

A standard curve was constructed using a set of taxol standard dilution.As a result, it was found that the supernatant contained 1.4 μg ofpaclitaxel from 1 liter of culture while the mycelium contained about160 ng of paclitaxel from 1 liter of culture.

EXAMPLE 12

Endogenous Paclitaxel Production Demonstration by Fermentation withRadioactive Precursors

A further evidence for production of paclitaxel by the strainKitasatosporia sp. CECT 4991 has been obtained in two feedingexperiments with radioactive precursors.

Feeding Experiment with L-[2,3,4,5,6-³H]phenylalanine

A frozen vial of the strain Kitasatosporia sp. CECT 4991 was defrostedand transferred in 300 mL Erlenmyer flask containing 30 mL of thefollowing liquid seed medium VS. The flask was incubated under aerobiccondition at 28° C. and 250 rpm. After 48 hours of incubation thelabeled precursor L-[2,3,4,5,6-³H]phenylalanine (120 μCi, specificactivity 5.11 TBq/mmol) was fed to the microorganism. After additional48 hours, the culture was centrifuged (30 minutes at 3000×g) and themycelium was extracted three times with MeOH. The organic solvent wasevaporated under reduced pressure and the aqueous residue was loadedonto two Extrelut 20 columns. The clear supernatant of the culture wasloaded onto three Extrelut 20 columns. Each column was extracted with120 mL of n-hexane (discarded), then with 80 mL of CH₂Cl₂, as previouslydescribed. The CH₂Cl₂ extracts were combined, dried under reducedpressure and to this crude extract of taxanes were added unlabeledBaccatin III and paclitaxel, 10 μg each. This extract was then subjectedto the chromatographic procedure of Table 5.

The incorporated radioactivity, even though rather low, is significantbecause the recovery of unlabeled baccatin allows exclusion of thepossibility that paclitaxel could be labeled by exchange with thetritiated water eventually present in the medium.

Feeding Experiment with ³H-Baccatin

A frozen vial of the strain Kitasatosporia sp. CECT 4991 was defrostedand transferred in 300 mL Erlenmyer flask containing 30 mL of thefollowing liquid seed medium VS. The flask was incubated under aerobiccondition for 48 hours at 28° C., 250 rpm. At completion of theincubation, 2 mL aliquot was used to inoculate aseptically a 300 mLErlenmyer flask containing 50 mL of the following liquid productivemedium PT. The flask was incubated under aerobic condition at 28° C.,250 rpm. After 24 hours of incubation the labeled precursor ³H-Baccatin(20 μCi, specific activity 1.83 MBq/mg) was fed to the microorganism.

After additional 144 hours, the culture was centrifuged (30 minutes at3000×g) and the clear supernatant was loaded onto two Extrelut columns.Each column was extracted with 100 mL of n-hexane (discarded), then with100 mL of CH₂Cl₂. The crude extract of taxanes was dried under reducedpressure, added with unlabelled paclitaxel, 50 μg, then submitted to thefollowing chromatographic procedure:

the crude CH₂Cl₂ extract was purified by chromatography on TLC in systema)

the band corresponding to paclitaxel was scraped, eluted andconcentrated

the extract of the band was submitted to a second chromatography insolvent b)

the band corresponding to paclitaxel was scraped, eluted andconcentrated

one half of the band containing paclitaxel was injected in HPLC and theradioactivity corresponding to peak of paclitaxel was determined byliquid scintillation.

the second half of the band was diluted to a theoretical concentrationof 125 μg/mL. The quantity of paclitaxel present in this solution wasdetermined by HPLC analysis in comparison with a calibration curve.

The radioactivity incorporated into the molecule of paclitaxel was 1.26nCi/50 mL of culture, corresponding to an incorporation of 510 ng/L ofbaccatin III and to a production of 742 ng/L of paclitaxel.

The recovery of the added unlabelled paclitaxel corresponding to 73%,the final estimated production of paclitaxel was of 942 ng/L.

HPLC Analysis

HPLC analysis were performed on a reversed-phase C18 column—5 μm-25×0.46cm using the following elution program:

from 0 to 20 minutes isocratic at 30% HPLC grade acetonitrile in Milli-Qgrade water,

from 20 to 25 minutes linear gradient from 30% to 50% acetonitrile inwater,

from 25 to 40 minutes isocratic at 50% acetonitrile in water.

The column was thermostated at 32° C. and eluted at a flow rate of 2mL/min.

A standard curve of paclitaxel concentration versus peak area was usedto quantify paclitaxel in samples.

EXAMPLE 13

Production of Paclitaxel in Fermenter

For the first stage of the seed phase four well-grown agar slants ofKitasatosporia sp. CECT 4991 were used to inoculate into eight 2 Lbaffled glass bottles containing 500 mL of the medium VS. For the secondstage of seed phase the baffled glass bottles were incubated at 28° C.,for 48 hours on a rotary shaker (130 rpm). At the end of incubation, theseed cultures obtained were pooled and 3.5 L of this pool weretransferred into a 200 L fermenter containing 100 L of the same mediaVS. The process was carried out at 28° C., for 48 hours, with agitationat 150 rpm remote control and air flow at a rate of 68 mL/min. A 15 Lportion of the vegetative culture by fermenter was inoculated into a 500L fermenter containing 300 L of the following productive media: morsuit25 g/L, dextrin 10 g/L, soytone 15 g/L, MOPS 5 g/L, K₂HPO₄ 0.5 g/L.After 120 hours of incubation, the mycelium was removed from the cultureby continuous centrifugation with a centrifugal separator and the clearsupernatant applied onto a XAD-2 column (30 L) previously washed withNaOH 1N (30 L), water (30 L), HCl (30 L), water (30 L), acetone (60 L),water (90 L).

After the adsorption, the resin was washed with 90 L of water(discarded) and eluted with ethanol (60 L). The ethanol extract wasconcentrated under reduced pressure to a volume of 10 L and extractedtwice with n-hexane (discarded) and twice with dichloremethane. Thedichloromethane crude extract was brought to dryness, re-suspended to a30 mL volume with methanol and partially purified according to theprocedure described by Mattina. M. J .I. and MacEachern. G. J. (J.Chrom. A 1994, 679, 269-275) using C18 disposable cartridges containing10 g of sorbent. Prior to use, the cartridge was conditioned with 100 mLethyl acetate, 100 mL methanol and 100 mL of Milli-Q grade water takingcare of leaving at least 1 cm of water above the top frit of thecartridge before loading an aliquot of 6 mL of the crude extract. Thecartridge was eluted with 50 mL of Milli-Q grade water, 100 mL of 20%methanol, 100 mL of 50% methanol, 100 mL of 80% methanol and 100 mL ofmethanol. The polyclonal antibody assay indicated that all taxanes hadselectively been eluted from the cartridge in the 80% methanol while themonoclonal antibody assay indicated a production of 2 μg/L of paclitaxelreferred to the original culture volume (300 L).

2 mL of the paclitaxel containing fraction were brought to dryness,resuspended in 20 μL of methanol and further purified by HPLC under theconditions described herein. Fractions were collected every minute fromthe outlet of the HPLC system; the resulting 40 fractions were monitoredfor paclitaxel content with the monoclonal antibody assay, as describedabove, that evidentiated paclitaxel in one single fraction eluted fromthe HPLC column between 27 and 28 minutes with an estimatedconcentration of 1.3 μg/L referred to the original culture volume (300L).

LC-MS Detection of Paclitaxel

The same sample was subjected to RP-HPLC using a 2.0×250 mm,pentafluorophenyl 5 μm column. Mobile phases A and B were respectively:

Phase A: Milli-Q grade water containing 0.1% of acetic acid glacial,99.99%

Phase B: acetonitrile HPLC grade containing 0.04% of acetic acidglacial, 99.99%.

Elutions were carried out with a steps gradient of B from 46.7% to 57.3%in 47 minutes at a flow rate of 0.165 mL/min. Separations were performedat room temperature and elution profiles were monitored with a diodearray detector at the wavelength of 227 nm.

On-line RP-HPLC/electrospray mass spectrometry was performed on a singlequadrupole instrument equipped with an electrospray interface.

Eluates from RP-HPLC were directly injected into the ion source of themass spectrometer. The electrospray potential was approx. 6 kV. Thequadrupole mass analyzer was set up to scan over a mass-to-charge ratio(m/z) from 150 to 920 Da, at 0.5 s per scan. The sum of data acquiredconstitued the final spectrum. Mass calibration was performed with amixture of valine, tri-tyrosine and hexa-tyrosine.

LC-MS analysis of this fraction confirmed the presence of paclitaxel byevidentiating a peak with the same RT (24.13 minutes, under theconditions reported in this section), UV spectrum and mass spectrum ofpaclitaxel. The concentration of paclitaxel determined by HPLC incomparison with a paclitaxel calibration curve, corresponded to 1 μg/Lreferred to the total volume of the culture (300 L).

TABLE 1 Method of Isolation Antibiotics Number of strains Example PlantSample pretreatment agar incorporated (mg/L) and related genus 1 Taxusinner IPIT 100% WA, Cycloheximide (60) 1 Streptomyces baccata bark CYApenicillin (10) 2 Actinoplanes 3 unidentified 2 Taxus inner IPIT 50% WA,ISP2 Cycloheximide (60) 1 Streptomyces baccata bark polimixin (5) 3Taxus bark IPIT 100% WA Cycloheximide (60) 6 Streptomyces baccata 1Nocardiopsis 1 Kitasatosporia 4 Taxus needles UV light WA Cycloheximide(60) 4 Streptomyces baccata nystatin (60) 8 Micromonospora tunicamicin(30) 1 unidentified 5 Taxus twigs propylenoxid WA, cycloheximide (60) 2Streptomyces brevifolia CYA nalidix acid (15) 1 Micromonospora 6 Torrejatwigs propylenoxid WA cycloheximide (60) 6 Streptomyces californicanystatin (60) 7 Taxus roots sodium WA, ISP2 cycloheximide (60) 4Streptomyces baccata hypochlorite tetracycline (10) 1 Actinomadura 8Taxus stem WA cycloheximide (60) 2 Streptomyces baccata tunicamicin (30)1 Actinomadura 9 Taxus bark UV light IPIT WA, cycloheximide (60) 20Streptomyces baccata 30% CYA novobiocin (25) 1 Nocardioforme 4unidentified WA = water agar; CYA = Czapek agar

TABLE 2 AERIAL SUBSTRATE PIG- MEDIA GROWTH MYCELIUM MYCELIUM MENT WA25++ gray colourless − ISP MEDIUM 2 +++ gray brown − ISP MEDIUM 3 +++ graygray − ISP MEDIUM 4 +++ gray beige − ISP MEDIUM 5 +++ gray light gray −ISP MEDIUM 7 +++ gray light gray − NUTRIENT +++ gray white − AGARBENNETT'S +++ gray beige − AGAR CZAPEK AGAR +++ gray gray −

TABLE 3 CARBOHYDRATE Glycerol + Erythritol − D-Arabinose − L-Arabinose −Ribose − D-Xylose + L-Xylose − Adonitol − β-Methyl-D-xyloside −Galactose + Glucose + Fructose − Mannose + Sorbose − Rhamnose − Dulcitol− Inositol − Mannitol − Sorbitol − α-Methyl-D-mannoside −α-Methyl-D-glucoside − N-Acetyl-glucosamine + Amygdalin + Arbutin +Esculin − Salicin ± Cellobiose + Maltose + Lactose − Melibiose −Sucrose + Trehalose + Inulin − Melezitose − Raffinose − Starch +Glycogen + Xylitol − Gentiobiose + D-Turanose − D-Lyxose − D-Tagatose −D-Fucose − L-Fucose − D-Arabitol − L-Arabitol − Gluconate + 2 Ketogluconate − 5 Keto gluconate −

TABLE 4 ENZYME ASSAYED FOR Phosphatase alcaline + Esterase (C 4) +Esterase Lipase (C 8) + Lipase (C 14) − Leucine arylamidase + Valinearylamidase − Cystine arylamidase − Trypsin − Chymotrypsin − Phosphataseacid + Naphthol-AS-B1-phpsphohydrolase + α-galactosidase −β-galactosidase + β-glucuronidase − α-glucosidase − β-glucosidase +N-acetyl-β-glucosaminidase − α-mannosidase − α-fucosidase −

TABLE 5 Purification procedure of the extract obtained from the cultureincubated with L-[2,3,4,5,6-³H]phenylalanine

TLC systems: a) TLC plates silica gel 60 (E. Merck, 64271 Darmstadt,Germany developed in n-hexane:acetone (1:1); b) TLC plates silica gel 60developed in # CH₃CN:CH₂Cl₂ (35:65). HPLC system: as described in theExample 12.

What is claimed is:
 1. A biologically pure culture of a microorganismbelonging to the genus Kitasatosporia, which produces a taxane.
 2. Themicroorganism according to claim 1 which produces a taxane selected fromthe group consisting of paclitaxel, 10 deacetyltaxol, 7-xylosyltaxol,7-epi-10-deacetyltaxol, 7-xylosyl-10-deacetyltaxol, 7-epi-taxol,7-epi-10-deacetylbaccatin III, baccatin III, 10-deacetyl baccatin III,cephalomannine and 10-deacetylcephalomannine.
 3. The microorganismaccording to claim 1 which produces paclitaxel.
 4. The microorganismaccording to claim 1 which produces a taxane which binds to an antibodyspecific for paclitaxel.
 5. The microorganism according to claim 1 whichproduces a taxane which binds to antibody R4, 8A10, 3C6 or 3H5.
 6. Abiologically pure culture of Kitasatosporia sp. CECT 4991.